Anti-IgE单链抗体纯化工艺研究及活性鉴定

目的:建立anti-IgE单链抗体纯化工艺并对其活性进行鉴定。方法:根据protein L亲和层析填料和Ni亲和层析填料的特点分析,经初筛后选用protein L填料作为第一步初纯化填料,Ni亲和层析填料作为第二步精细纯化填料。通过对这两种纯化方式的上样条件和洗脱条件进行研究,建立了anti-IgE单链抗体的纯化工艺。结果:protein L亲和层析的初纯化工艺:最佳杂质洗涤pH=3.5,最佳目标蛋白洗脱pH=2.0,并且pH=2.0洗脱的目标蛋白收集在第二步Ni亲和层析的上样缓冲液中,可直接进行第二步Ni亲和层析纯化。所建立的Ni亲和层析精细纯化工艺:最佳杂质洗涤为50mmol/L咪唑,最佳目标蛋白洗脱为500mmol/L咪唑。经两步亲和纯化,目标产物在SDS-PAGE上纯度为99.0%,CE-SDS上纯度为99.5%,两步总收率为80.0%。纯化后的蛋白经竞争ELISA和Biacore检测,证实该产品特异性识别IgE靶标,与IgE靶标的亲和力达到8.59e~(-9)M,并且竞争Biacore结果显示该抗体对IgE有良好的中和活性,其抑制IgE的EC50值为70n M。结论:建立了一种高效、简洁的大肠杆菌表达的anti-IgE单链抗体纯化工艺,为其进一步规模放大工艺的建立奠定了基础。同时证明了该新型小分子anti-IgE抗体对靶标具有良好的特异性、亲和力以及中和活性,并展示出其在医用中的应用价值。所建立的小分子抗体纯化工艺技术对其他小分子单链抗体的纯化具有参考价值。     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

大孔吸附树脂纯化亚麻木酚素

以纯化亚麻木酚素粗品为目的,比较了X-5、D101和AB-8三种大孔吸附树脂对木酚素的吸附性能,筛选出X-5大孔树脂最合适。通过动态吸附、解吸实验确定了上样浓度与上样量。在上样浓度10 mg/m L,上样量25 m L时分离效果最佳。同时,重点考察了梯度洗脱和等度洗脱两种解吸方式对木酚素产品纯度和收率的影响。等度洗脱采用30%乙醇水溶液,木酚素纯度可达85.49%,收率为57.59%;采用60%乙醇水溶液洗脱,木酚素纯度为59.30%,收率最高为75.05%。梯度洗脱可得到不同纯度产品,过程总收率为77.63%。     

桑椹中黄酮类物质的大孔树脂纯化工艺

以桑椹中黄酮类物质的吸附量和解吸率为指标,对比分析HZ-801、HZ-816、HZ-818等12种大孔吸附树脂对桑椹提取液的分离纯化效果,优选出最佳树脂HZ-801并通过对上样液pH、上样液质量浓度、上样量、吸附流速、洗脱剂质量浓度、洗脱剂用量、洗脱流速等影响因素的考察,确定最优工艺:吸附阶段上样液pH=4,上样液质量浓度0.45mg/mL,上样量420mL,吸附流速120mL/h,动态吸附量(干树脂)25.34mg/g,吸附率84.25%;洗脱阶段的洗脱剂体积分数为60%乙醇,洗脱剂用量270mL,洗脱流速120mL/h。此优化工艺条件下的洗脱率为85.78%,总黄酮纯度从23.64%提高到82.36%。     

离子交换层析结合反向色谱纯化聚二氨基丙酸

ε-聚赖氨酸生产菌株Streptomyces albulus PD-1可合成一种新型非蛋白质氨基酸均聚物聚二氨基丙酸,采用离子交换层析和反向色谱,对聚二氨基丙酸的分离纯化进行研究。离子交换层析柱选用DEAE-Sepharose Fast Flow填料,50 mmol/L磷酸盐缓冲液(p H 7.5)平衡上样,含0.5 mol/L Na Cl的磷酸盐缓冲液(p H 7.5)洗脱,收集洗脱液用分子筛Sephadex G-25除去磷酸盐缓冲液。然后用C18反相色谱进一步纯化,流动相为V(甲醇)/V(0.1%磷酸)=5/95。经过离子交换层析和反向色谱,纯化得到聚二氨基丙酸纯品,回收率为39.8%,样品纯度达98.4%,为后续的聚二氨基丙酸的深入研究奠定基础。     

星点设计-效应面法优化大孔树脂纯化还原型萝卜硫苷工艺及体外抗氧化活性研究

为研究大孔树脂纯化还原型萝卜硫苷的最佳工艺及其抗氧化能力。实验以8种不同型号的大孔树脂对还原型萝卜硫苷的比吸附量、吸附率和洗脱率为指标筛选出最佳型号的大孔树脂,采用单因素考察和星点设计-效应面法优选出大孔树脂纯化还原型萝卜硫苷的工艺参数,通过测定纯化前后还原型萝卜硫苷提取物对DPPH自由基及ABTS~+·自由基的清除能力来表征其抗氧化活性。结果表明,HPD-722型大孔树脂纯化还原型萝卜硫苷效果最好,最佳纯化工艺为:上样液pH 5.3,上样流速2.5 BV/h,上样液浓度0.53 mg/mL;洗脱液为70%乙醇溶液,洗脱液体积为2.5 BV,洗脱液流速为1.5 BV/h,还原型萝卜硫苷纯度由0.404%提高到17.903%,纯度提高了44.35倍,纯化后的还原型萝卜硫苷GRH提取物与萝卜提取液相比,清除DPPH自由基和ABTS~+·自由基的能力分别提高了67.31和45.27倍。     

老熟莲子壳多酚的树脂法纯化及稳定性研究

通过吸附和解吸实验确定柱层析分离和纯化莲子壳多酚的大孔树脂及工艺参数,并对莲子壳多酚的稳定性进行研究。结果表明:AB-8树脂最适合于莲子壳多酚的分离纯化,最佳工艺条件为:上样浓度4.5 mg/mL,上样速度0.5 mL/min,水洗至无糖后,以50%的乙醇洗脱,洗脱流速2 mL/min,洗脱体积4 BV,在此条件下,莲子壳多酚纯度达69.34%。莲子壳多酚的稳定性受光、温度和pH影响,温度越高,光照越强,碱性越强,降解程度越大。Fe3+、Fe2+、Al3+、Sn2+、Cu2+、Pb2+这6种离子对莲子壳多酚具有显著的破坏作用。     

宁夏枸杞叶总黄酮提取和纯化方法的优化

采用L9(3⁴)正交设计,以黄酮含量为指标,确定的宁夏枸杞叶总黄酮的最佳超声提取方法为: 30倍70%乙醇超声提取50 min,超声功率为250 W;纯化方法条件为: 5倍柱体积70%乙醇洗脱,浓度为4 mg/mL、pH值5.0的上样液,上样速度和洗脱流速均为1 mL/min。并以吸附率、解吸率、回收率等作为考察参数,对提取和纯化方法进行了验证。实验结果表明,该方法简便节时、易操作且毒害小,各项变异系数低。     

正交试验法优化加杨树皮中白杨素的纯化工艺

以白杨素纯度为指标,采用L_(9 )(3~4)正交试验法,考察聚酰胺柱层析的洗脱溶剂、上样浓度、上样量及流速对杨树皮中白杨素纯度的影响,优化聚酰胺纯化工艺。最佳工艺条件为:以60-80目的聚酰胺装柱,以60%乙醇为流动相,上样浓度为稀释3倍的经石油醚冷浸脱脂后的杨树皮总酚酸流浸膏,上样量与聚酰胺柱的体积比为1∶2,吸附流速20 ml/min,洗脱流速为80 ml/min。收集60%乙醇溶液洗脱部位,经重结晶可得白杨素粗品。该纯化工艺简单、合理,是杨树皮中白杨素的最佳纯化工艺。     

疏水层析分离正确折叠与错误折叠的复合干扰素

采用疏水层析纯化重组复合干扰素,成功去除了复性过程中产生的错误折叠体、聚集体及杂蛋白,并考察了配基类型、盐浓度、pH值和流速对疏水层析纯化效果的影响,结果表明采用ButylSepharose 4FastFlow、硫酸铵初始浓度0.8mol/L、缓冲液pH值8.3、线流速为90cm h时疏水层析纯化效果最佳,最终目标蛋白反相高效液相色谱检测纯度达到99.6% ,还原及非还原型SDS PAGE电泳均呈单一条带,其比活为2.3×10⁹IU/mg,回收率为36.7%。     

Expanded bed adsorption as a unique unit operation for the isolation of bacteriocins from fermentation media

Expanded bed adsorption using a strong cation exchanger allowed the direct isolation of amylovorin L471, a bacteriocin from Lactobacillus amylovorusDCE 471, from the fermentation medium. The pH of the loading and elution buffer were optimised in a packed bed with cell-free culture supernatant. Bound bacteriocin was eluted with 1.0 M NaCl. The highest recovery (30%) was obtained at the lowest pH (3.6). At higher pH values the recovery was lower, namely 12%, 15% and 7% at pH 4.5, 6.5 and 8.0, respectively. In expanded bed mode, direct isolation of the bacteriocin from the fermentation medium at pH 3.6 (loading and elution) initially resulted in a recovery of 12%. After optimisation of the pH (loading and elution at pH 3.6 and 6.5, respectively), the recovery for amylovorin L471 increased up to 30% and higher. Recovery of enterocin A from Enterococcus faeciumCTC 492 fermentation medium averaged 15% (loading and elution at pH 3.6 and 6.0, respectively). With pediocin, produced by Pediococcus acidilactici ATCC 8042, 26% recovery was obtained at a pH of 6.5 during loading and elution. Low recoveries can be ascribed to non-optimal operation conditions (pH of loading and elution buffer), inactivation of the bacteriocin on a cationic resin, and the formation of more insoluble and less active, strongly hydrophobic bacteriocin aggregates upon further purification.     

Expanded bed adsorption: elution in expanded bed mode

Elution in expanded bed mode has been investigated in the expanded bed adsorption process. Elution was performed at different sample loads and at different liquid velocities using bovine serum albumin as a model. The effect on mixing in the liquid phase and on the volume of the eluted peak were determined. Mixing in the liquid phase was almost unaffected when elution was performed at 100 cm/h, regardless of sample load. However, mixing increased significantly when elution was carried out at high liquid velocities (300 cm/h) at high sample loads. The eluted peak volume increased with liquid velocity and increased sample load. It was approx. 80% higher in expanded bed mode than in packed bed from an adsorbent completely saturated with protein eluted at 300 cm/h.     

小鼠肠道菌群HPLC分析中色谱分离条件的优化

研究了在小鼠肠道菌群的高效离子交换色谱（HPLC）分析中,不同色谱分离条件对分离效果的影响,确立了最佳色谱条件：样品上样于Toyopearl TSKgel SuperQ-650c强阴离子交换树脂柱,以0.02mol/L哌嗪-HCl缓冲液（pH8.0）平衡,洗脱盐浓度为1.0mol/L NaCl,洗脱梯度为0.1-0.5mol/L NaCl线性梯度洗脱80min,再经0.5-0.75mol/L NaCl线性梯度洗脱25min,流速1mL/min,进样量为1mL。小鼠肠道菌群HPLC分析方法的建立,为深入研究小鼠肠道菌群的组成和动态变化奠定了基础。     

Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: comparison between polymer brush coating and surface functionalization

In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe(3+), were prepared by in situ chemical modification of fused silica capillary using two chemistries, polymer brush coating and surface functionalization. One column was based on a poly-(glycidyl methacrylate) brush (GMA) and the other on 3-glycidoxypropyltrimethoxysilane (GLYMO). Phosphopeptide enrichment on the open tubular columns was evaluated on an α(S1), α(S2) mixture and β casein peptides. The optimized enrichment protocol includes sample loading in a slightly acidic solution made with pure deionized water, a washing step with 10% acetonitrile, 0.1% formic acid, and an elution step with 50% acetonitrile, 0.1% phosphoric acid at pH 8.0. MALDI-TOF spectra generated from eluted fractions show several phosphorylated peptides. For example, 7 phosphorylated peptides of the α(S1), α(S2) casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe(3+) IMAC polymer brush column elutes all phosphorylated peptides in one fraction independently of phosphorylation degree, whereas the GLYMO-IMAC polymer brush provides longer elution times for higher phosphorylation states. In particular, the pentaphosphorylated peptide was eluted after a 30 min elution versus 5 min for monophosphorylated species (isocratic gradient).     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

Affinity enrichment of bovine lactoferrin in whey

Bovine lactoferrin was enriched in various whey samples by affinity chromatography using immobilized gangliosides. Bovine gangliosides were isolated from fresh buttermilk using a combination of ultrafiltration and organic extraction. Isolated gangliosides were covalently immobilized onto controlled-pore glass beads. The immobilized matrix contained 66 micrograms of gangliosides per gram of beads. After loading the matrix with reconstituted whey protein isolate (WPI) or whey protein concentrate (WPC), the matrix was washed with sodium phosphate buffer (pH 7) followed by sodium acetate buffer (pH 4) before elution of lactoferrin with 1 M NaCl in sodium acetate buffer. From the intensities of the protein bands in SDS-PAGE, lactoferrin constituted a minimum of 40% of the total protein in the salt eluted sample. WPI, pretrated by heating and ultrafiltration, showed the highest lactoferrin purity among protein sources, while WPI (10% wt/vol) showed the highest recovery. These results show that immobilized gangliosides can be used to enrich the lactoferrin content of whey.     

Concentration of viruses from environmental waters using nanoalumina fiber filters

Human viral contamination in drinking and recreational water may persist for extensive periods of time and cause a significant health risk concern. The aim of this study is to evaluate a viral recovery method using a new electropositive charged nanoalumina filter and to compare results with the widely used negatively charged HAWP filter by Millipore Inc. The recovery of infectious recombinant adenovirus type 5 (rAd5) was tested using the Fluorescence-Activated Cell Sorting (FACS) assay, in parallel with viral genomes recovery assay by quantitative PCR (qPCR). The mean infectivity recoveries were 82-91% by nanoalumina filters eluted with 3% beef extract (BE, pH6.0), and 78-90% by HAWP filters eluted with 3% BE (pH 9.0), respectively, from 1 L of environmental samples seeded with 1pfu/mL rAd5. The mean genome recoveries were 16-35% by nanoalumina filters eluted with BE (pH 6.0), and 29-66% by HAWP filters eluted with NaOH (pH 10.8) from different types of water, respectively. Water quality, concentration of viruses, filters, and elution buffers are factors that determine the viral recovery efficiencies. The nanoalumina filters also had higher filtration rates than HAWP filters for large volumes of environmental water samples (up to10 L), thus, have an advantage in concentrating infectious viruses from environments without pre-filtration, adjusting pH or adding multivalent cations.     

Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody

Active eukaryotic RNA polymerase II (RNAP II) was purified by immunoaffinity chromatography, using a monoclonal antibody (mAb) that reacts with the highly conserved heptapeptide repeat of the largest subunit. This mAb (designated SWG16) was conjugated to CNBr-activated Sepharose and used to purify RNAP II from wheat germ and calf thymus. The subunit composition of the immunoaffinity-purified enzyme was essentially the same as RNAP II purified by conventional chromatography except that it contained only the form with the unproteolyzed largest subunit. Active enzyme could be eluted from the SWG16-Sepharose, at pH 7.9, with combinations of low molecular weight polyols and nonchaotropic salts. The superior eluting procedure used combinations of ethylene glycol (30-40%) and ammonium sulfate (0.5-0.75 M). Active enzyme also could be eluted with a synthetic peptide containing four repeats of the heptapeptide; however, the peptide was not as effective as the polyol and salt combinations for eluting the enzyme. This mAb should be useful for purifying RNAP II from many eukaryotic species. Because the elution of enzyme from the immunoadsorbent seems to be dependent upon the presence of a polyol, this antibody is referred to as a "polyol-responsive mAb." A procedure that helps to identify a polyol-responsive mAb and to optimize the eluting conditions is described. Polyol-responsive mAbs might have broad applicability to the purification of many labile enzymes by immunoaffinity chromatography.     

Analytical grade C-phycocyanin obtained by a single-step purification process

C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step.